Field of the Invention
The present invention generally relates to the detection and quantification of particles in a fluid sample, and more specifically relates to a method and device for detecting bacteria and determining the concentration thereof in a liquid sample and, in particular, a urine sample.
Description of the Prior Art
A number of methods are conventionally used to detect and evaluate bacteria in a urine sample. For example, there exist automated analyzers for use in evaluating urine sediment, which mostly utilize flowing a liquid sample through a flow cell and employing either flow cytometry or image analysis of the flowing particles. There are also different types of fluid image capture methods that may be performed, including the optical sectioning methods disclosed in U.S. Pat. No. 8,780,181 (Olesen, et al.) and U.S. Patent Application Publication No. 2012/0244519 (Olesen, et al.).
Alternatively, another conventional method for the detection and evaluation of bacteria in a urine sample involves the manual observations conducted by medical technicians using bright field microscopy. More specifically, this standard method for urine microscopy includes spinning a liquid sample in a centrifuge and discarding the supernatant, leaving only a sediment pellet. The pellet is then re-suspended and evaluated on a microscope slide under a cover slip using a microscope. With this method, the fluid depth is very shallow. For a 30 microliter aliquot with a conventional 22×22 millimeter cover slip, the depth will be approximately 60 microns, and spacing is confined to a more two dimensional space than the three dimensional volume provided by a deeper fluid channel. Such methods, of course, are time consuming and tedious for the medical technician, and often lead to erroneous results in quantifying the bacteria present in the sample due to the small size of the bacteria and limitations of bright field microscopy.
Urine sediment analysis using imaging techniques must detect bacteria in a urine sample in the presence of small non-bacteria debris. This requirement poses challenges, since bacteria are approximately one micron in size, which is near the limit of detection of air-coupled bright field microscopic imaging techniques. With this restriction, bacteria can be seen, but geometric properties cannot be determined, since each bacterium may be represented by only a single pixel due to its size. This limitation makes it difficult to determine the difference between bacteria and small debris (non-bacteria) particles. This difficulty is also present in standard bright field microscopy, where it can be difficult for a technician to identify a specific particle as bacteria or not, even with 400× magnification. There are other techniques that are used, including evaluating the uniformity of particle sizes and positions within the fluid, as well as colony formations that are indicative of bacteria. If bacterial presence is to be confirmed, then alternate techniques such as dry, stained slides that are evaluated under bright field microscopy or quantitative culture are employed to confirm the presence of bacteria.
The difficulties described above with conventional techniques call for a more reliable bacteria detection technique in a debris-filled urine environment.